Reduced OxyR positively regulates the prodigiosin biosynthesis in Serratia marcescens FS14.

OxyR, a LysR family transcriptional regulator, plays vital roles in bacterial oxidative stress response. In this study, we found that the deletion of oxyR not only inhibited the antioxidant capacity of S. marcescens FS14, but also decreased the production of prodigiosin. Further study revealed that OxyR activated the prodigiosin biosynthesis at the transcriptional level. Complementary results showed that not only the wild-type OxyR but also the reduced form OxyRC199S could activate the prodigiosin biosynthesis. We further demonstrated that reduced form of wild type OxyR could bind to the promoter of pig gene cluster, and identified the binding sites which is different from oxidized OxyR binding sites in E. coli. Our results demonstrated that OxyR in FS14 uses oxidized form to regulate the expression of the antioxidant related genes and utilizes reduced form to activate prodigiosin production. Further in silico analysis suggested that the activation of prodigiosin biosynthesis by reduced OxyR should be general in S. marcesencs. To our knowledge, this is the first report to show that OxyR uses the reduced form to activate the gene's expression, therefore, our results provide a novel regulation mechanism of OxyR.

Structural insight into subunit F of respiratory chain complex I from Xanthomonas oryzae pv. oryzae inhibition by parthenolide.

BACKGROUND: Bacterial leaf blight caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the most serious diseases of rice, and there is a lack of bactericides for controlling this disease. We previously found parthenolide (PTL) is a potential lead for developing bactericides against Xoo, and subunit F of respiratory chain complex I (NuoF) is an important target protein of PTL. However, the binding modes of PTL with NuoF need further elucidation. RESULTS: In this study, we obtained the crystal structure of Xoo NuoEF (complex of subunit E and F of respiratory chain complex I) with a resolution of 2.36 Å, which is the first report on the protein structure of NuoEF in plant-pathogenic bacteria. The possible binding sites of PTL with NuoF (Cys105 and Cys187) were predicted with molecular docking and mutated into alanine using a base mismatch method. The mutated proteins were expressed in Escherichia coli and purified with affinity chromatography. The binding abilities of PTL with mutated proteins were investigated via pull-down assay and BIAcore analysis, which revealed that double mutation of Cys105 and Cys187 in NuoF severely affected the binding ability of PTL with NuoF. In addition, the binding modes were further simulated with combined quantum mechanical/molecular mechanical calculations, and the results indicated that PTL may have a stronger binding with Cys105 than Cys187. CONCLUSION: NuoEF protein structure of Xoo was resolved, and Cys105 and Cys187 in NuoF are important binding sites of PTL. This study further clarified the action mechanism of PTL against Xoo, and will promote the innovation of bactericides targeting Xoo complex I. © 2024 Society of Chemical Industry.

Multicopy expression of sigma factor RpoH reduces prodigiosin biosynthesis in Serratia marcescens FS14.

Regulation of prodigiosin biosynthesis is received wide attention due to the antimicrobial, immunosuppressive and anticancer activities of prodigiosin. Here, we constructed a transposon mutant library in S. marcescens FS14 to identify genes involved in the regulation of prodigiosin biosynthesis. 62 strains with apparently different colors were obtained. Identification of the transposon insertion sites revealed that they are classified into three groups: the coding region of cyaA and two component system eepS/R and the promoter region of rpoH. Since the effect of cyaA and eepS/R genes on prodigiosin was extensively investigated in Serratia marcescens, we chose the mutant of rpoH for further investigation. Further deletion mutation of rpoH gene showed no effect on prodigiosin production suggesting that the effect on prodigiosin production caused by transposon insertion is not due to the deletion of RpoH. We further demonstrated that multicopy expression of RpoH reduced prodigiosin biosynthesis indicating that transposon insertion caused RpoH enhanced expression. Previous results indicate that RpoS is the sigma factor for transcription of pig gene cluster in FS14, to test whether the enhanced expression of RpoH prevents prodigiosin by competing with RpoS, we found that multicopy expression of RpoS could alleviate the prodigiosin production inhibition by enhanced RpoH. We proposed that multicopy expressed RpoH competes with RpoS for core RNA polymerase (RNAP) resulting in decreased transcription of pig gene cluster and prodigiosin production reduction. We also demonstrated that RpoH is not directly involved in prodigiosin biosynthesis. Our results suggest that manipulating the transcription level of sigma factors may be applied to regulate the production of secondary metabolites.

Crystal structures of herbicide-detoxifying esterase reveal a lid loop affecting substrate binding and activity.

SulE, an esterase, which detoxifies a variety of sulfonylurea herbicides through de-esterification, provides an attractive approach to remove environmental sulfonylurea herbicides and develop herbicide-tolerant crops. Here, we determined the crystal structures of SulE and an activity improved mutant P44R. Structural analysis revealed that SulE is a dimer with spacious binding pocket accommodating the large sulfonylureas substrate. Particularly, SulE contains a protruding ß hairpin with a lid loop covering the active site of the other subunit of the dimer. The lid loop participates in substrate recognition and binding. P44R mutation altered the lid loop flexibility, resulting in the sulfonylurea heterocyclic ring repositioning to a relative stable conformation thus leading to dramatically increased activity. Our work provides important insights into the molecular mechanism of SulE, and establish a solid foundation for further improving the enzyme activity to various sulfonylurea herbicides through rational design.

A link between STK signalling and capsular polysaccharide synthesis in Streptococcus suis.

Synthesis of capsular polysaccharide (CPS), an important virulence factor of pathogenic bacteria, is modulated by the CpsBCD phosphoregulatory system in Streptococcus. Serine/threonine kinases (STKs, e.g. Stk1) can also regulate CPS synthesis, but the underlying mechanisms are unclear. Here, we identify a protein (CcpS) that is phosphorylated by Stk1 and modulates the activity of phosphatase CpsB in Streptococcus suis, thus linking Stk1 to CPS synthesis. The crystal structure of CcpS shows an intrinsically disordered region at its N-terminus, including two threonine residues that are phosphorylated by Stk1. The activity of phosphatase CpsB is inhibited when bound to non-phosphorylated CcpS. Thus, CcpS modulates the activity of phosphatase CpsB thereby altering CpsD phosphorylation, which in turn modulates the expression of the Wzx-Wzy pathway and thus CPS production.

Structural basis for substrate binding and catalytic mechanism of the key enzyme VioD in the violacein synthesis pathway.

Violacein is a pigment synthesized by gram-negative bacteria with various biological activities such as antimicrobial, antiviral, and anticancer activities. VioD is a key oxygenase converting protodeoxyviolaceinic acid to protoviolaceinic acid in violacein biosynthesis. To elucidate the catalytic mechanism of VioD, here, we resolved two crystal structures of VioD, a binary complex structure containing VioD and a FAD and a ternary complex structure composed of VioD, a FAD and a 2-ethyl-1-hexanol (EHN). Structural analysis revealed a deep funnel like binding pocket with wide entrance, this pocket is positively charged. The EHN is located at the deep bottom of the binding pocket near isoalloxazine ring. Further docking simulation help us to propose the mechanism of the hydroxylation of the substrate catalyzed by VioD. Bioinformatic analysis suggested and emphasized the importance of the conserved residues involved in substrate binding. Our results provide a structural basis for the catalytic mechanism of VioD.

Target Density Effects on Charge Transfer of Laser-Accelerated Carbon Ions in Dense Plasma.

We report on charge state measurements of laser-accelerated carbon ions in the energy range of several MeV penetrating a dense partially ionized plasma. The plasma was generated by irradiation of a foam target with laser-induced hohlraum radiation in the soft x-ray regime. We use the tricellulose acetate (C_{9}H_{16}O_{8}) foam of 2 mg/cm^{3} density and 1 mm interaction length as target material. This kind of plasma is advantageous for high-precision measurements, due to good uniformity and long lifetime compared to the ion pulse length and the interaction duration. We diagnose the plasma parameters to be T_{e}=17 eV and n_{e}=4×10^{20} cm^{-3}. We observe the average charge states passing through the plasma to be higher than those predicted by the commonly used semiempirical formula. Through solving the rate equations, we attribute the enhancement to the target density effects, which will increase the ionization rates on one hand and reduce the electron capture rates on the other hand. The underlying physics is actually the balancing of the lifetime of excited states versus the collisional frequency. In previous measurement with partially ionized plasma from gas discharge and z pinch to laser direct irradiation, no target density effects were ever demonstrated. For the first time, we are able to experimentally prove that target density effects start to play a significant role in plasma near the critical density of Nd-glass laser radiation. The finding is important for heavy ion beam driven high-energy-density physics and fast ignitions. The method provides a new approach to precisely address the beam-plasma interaction issues with high-intensity short-pulse lasers in dense plasma regimes.

Bioaugmentation with Tetrasphaera to improve biological phosphorus removal from anaerobic digestate of swine wastewater.

Tetrasphaera-enhanced biological phosphorus removal (T-EBPR) was developed by augmenting conventional EBPR (C-EBPR) with Tetrasphaera to improve phosphorus removal from anaerobic digestate of swine wastewater. At influent total phosphorus (TP) concentrations of 45-55 mg/L, T-EBPR achieved effluent TP concentration of 4.17 ± 1.02 mg/L, 54 % lower than that in C-EBPR (8.98 ± 0.76 mg/L). The enhanced phosphorous removal was presumably due to the synergistic effect of Candidatus Accumulibacter and Tetrasphaera occupying different ecological niches. Bioaugmentation with Tetrasphaera promoted the polyphosphate accumulation metabolism depending more on the glycolysis pathway, as evidenced by an increase in intracellular storage compounds of glycogen and polyhydroxyalkanoates by 0.87 and 0.34 mmol C/L, respectively. The enhanced intracellular storage capacity was coincidentally linked to the increase in phosphorus release and uptake rates by 1.23 and 1.01 times, respectively. These results suggest bioaugmentation with Tetrasphaera could be an efficient way for improved phosphorus removal from high-strength wastewater.

BarA/UvrY differentially regulates prodigiosin biosynthesis and swarming motility in Serratia marcescens FS14.

BarA/UvrY, a two-component system and global regulator that controls expression of more than a hundred of genes involved in virulence, motility, biofilm formation, and central carbon metabolism under various stress conditions. In this study, we investigated the function of BarA/UvrY system in Serratia marcescens FS14. The disruption of barA or/and uvrY results in the yield increase of secondary metabolite prodigiosin. We further demonstrated that BarA/UvrY system represses prodigiosin production by inhibiting the transcription level of pig gene cluster with direct binding to the pigA promoter. In addition, deletion of barA or/and uvrY abolished the swarming motility of FS14, but not the swimming motility. We revealed that BarA/UvrY activates swarming through directly upregulating the expression of the biosurfactant synthesis gene swrW rather than flagella system. We also observed that BarA/UvrY positively regulates the resistance to H2O2 same as in Escherichia coli highlighting the importance of BarA/UvrY on hydrogen peroxide resistance. Our results demonstrated that the BarA/UvrY system differentially regulates the biosynthesis of the secondary metabolite prodigiosin and swarming motility in S. marcescens FS14. Comparison of our results with those observed for Serratia sp. 39006 suggests that BarA/UvrY's role in regulation of secondary metabolite production is different among Serratia species.

Crystal structure of prodigiosin binding protein PgbP, a GNAT family protein, in Serratia marcescens FS14.

Acetylation is a conserved modification catalyzed by acetyltransferases that play prominent roles in a large number of biological processes. Members of the general control non-repressible 5 (GCN5)-N-acetyltransferase (GNAT) protein superfamily are widespread in all kingdoms of life and are characterized by highly conserved catalytic fold, and can acetylate a wide range of substrates. Although the structures and functions of numerous eukaryotic GNATs have been identified thus far, many GNATs in microorganisms remain structurally and functionally undescribed. Here, we determined the crystal structure of the putative GCN5-N-acetyltransferase PgbP in complex with CoA in Serratia marcescens FS14. Structural analysis revealed that the PgbP dimer has two cavities, each of which binds a CoA molecule via conserved motifs of the GNAT family. In addition, the biochemical studies showed that PgbP is a prodigiosin-binding protein with high thermal stability. To our knowledge, this is the first view of GNAT binding to secondary metabolites and it is also the first report of prodigiosin binding protein. Molecular docking and mutation experiments indicated that prodigiosin binds to the substrate binding site of PgbP. The structure-function analyses presented here broaden our understanding of the multifunctionality of GNAT family members and may infer the mechanism of the multiple biological activities of prodigiosin.

Exploration of Spatio-Temporal Characteristics of Carbon Emissions from Energy Consumption and Their Driving Factors: A Case Analysis of the Yangtze River Delta, China.

For the Yangtze River Delta (YRD) region of China, exploring the spatio-temporal characteristics of carbon emissions from energy consumption (CEECs) and their influencing factors is crucial to achieving carbon peaking and carbon neutrality as soon as possible. In this study, an improved LMDI decomposition model based on the Tapio model and Kaya's equation was proposed. Combined with the improved LMDI and k-means cluster analysis methods, the energy structure, energy intensity, unit industrial output value and population size were selected as the driving factors, and the contribution of each driving factor to the CEECs of prefecture-level cities was quantitatively analyzed. Our study found that: (1) By 2020, the total amount of CEECs in the 26 prefecture-level cities in the YRD will stabilize, while their intensity has shown a downward trend in recent years. (2) The decoupling relationship between CEECs and economic development generally showed a trend from negative decoupling to decoupling. The dominant factor in decoupling was generally the shift of DEL values towards urbanization rate and energy intensity and the open utilization of energy technologies. (3) From 2000 to 2010, the dominant factors affecting CEECs in 26 cities were energy intensity and energy structure, followed by industrial output value and urbanization rate. In general, the promotion effect of economic development on carbon emissions in the YRD region was greater than the inhibitory effect. After 2010, the restrictive effect of various factors on CEECs increased significantly, among which the role of gross industrial output was crucial. The research results can provide a scientific policy basis for the subsequent spatial management and control of carbon emission reduction and carbon neutrality in the YRD region at a finer scale.

Differentiated strategies of animal-derived and plant-derived biochar to reduce nitrogen loss during paper mill sludge composting.

This work aimed to reveal the differences of nitrogen (N) transformation between animal-derived and plant-derived biochar during paper mill sludge composting. Three treatments were established, including CK (no biochar), ABC (animal-derived biochar), and PBC (plant-derived biochar). Results showed that N loss was reduced by 24.43% and 35.50% in ABC and PBC, respectively, compared with CK. Moreover, the contents of acid-insoluble N (AIN) in ABC and bioavailable organic N (BON) in PBC were 6.180 g/kg and 9.269 g/kg higher than in CK (2.602 g/kg and 8.988 g/kg). The protease activity and bacterial abundance associated with the generation of humic N-containing precursors increased in ABC. Low urease activity and a more complex bacterial N-cycling network were found in PBC. Structural equation model confirmed that AIN formation and BON retention were the dominant strategies for animal-derived and plant-derived biochar, respectively. The findings provided multiple pathways to produce N-enriched compost products.

Linking Microbial Community Succession With Substance Transformation in a Thermophilic Ectopic Fermentation System.

Ectopic fermentation system (EFS) is an effective technology for treating mass livestock manure. However, the associations between microbial communities and substance transformation remain controversial. This study aimed to investigate chicken manure EFS lasting 170 days using 16S rRNA sequencing and electrochemical, spectroscopic, and chromatographic analyses. The results showed a noticeable transformation of protein-like substances into humus-like substances. Meanwhile, the electron-accepting capacity increased persistently, effectively reflecting the humification of organic substances. The contents of phenols that promoted electron transfer continued to increase from 2.80 to 6.00%, which could be used as a maturity indicator for EFS. During the heating period, the dominant microbial communities were Chloroflexi and Proteobacteria, whereas thermotolerant bacteria Cyanobacteria and Planctomycetes were significantly enriched from 1.64 to 50.15% during the continuous thermophilic period of EFS. The correlation analysis manifested that these thermotolerant bacteria were the major functional bacteria for the formation of phenols and the key to driving the humification of organic substances. This study provides insights into understanding the humification mechanisms and implementing regulatory strategies in EFS.

Crystal structures of PigF, an O-methyltransferase involved in the prodigiosin synthetic pathway, reveal an induced-fit substrate-recognition mechanism.

Prodigiosin, a red linear tripyrrole pigment, is a typical secondary metabolite with numerous biological functions, such as anticancer, antibacterial and immunosuppressant activities, and is synthesized through a bifurcated biosynthesis pathway from 4-methoxy-2,2'-bipyrrole-5-carbaldehyde (MBC) and 2-methyl-3-n-amylpyrrole (MAP). The last step in the biosynthetic pathway of MBC is catalysed by PigF, which transfers a methyl group to 4-hydroxy-2,20-bipyrrole-5-carbaldehyde (HBC) to form the final product MBC. However, the catalytic mechanism of PigF is still elusive. In this study, crystal structures of apo PigF and S-adenosylhomocysteine (SAH)-bound PigF were determined. PigF forms a homodimer and each monomer consists of two domains: a C-terminal catalytic domain and an N-terminal dimerization domain. Apo PigF adopts an open conformation, while the structure of the complex with the product SAH adopts a closed conformation. The binding of SAH induces dramatic conformational changes of PigF, suggesting an induced-fit substrate-binding mechanism. Further structural comparison suggests that this induced-fit substrate-recognition mechanism may generally exist in O-methyltransferases. Docking and mutation studies identified three key residues (His98, His247 and Asp248) that are crucial for enzyme activity. The essential function of His247 and Asp248 and structure analysis suggests that both residues are involved in activation of the HBC substrate of PigF. The invariance of Asp248 in PigF further confirmed its essential role. The invariance and essential role of His98 in PigF suggests that it is involved in correctly positioning the substrate. This study provides new insight into the catalytic mechanism of PigF, reveals an induced-fit substrate-recognition model for PigF and broadens the understanding of O-methyltransferases.

Crystal structures of TTHA1265 and TTHA1264/TTHA1265 complex reveal an intrinsic heterodimeric assembly.

Zinc peptidase M16 family members are widely distributed in most prokaryotic and eukaryotic organisms. M16 family has been divided into three subfamilies, M16A, M16B and M16C, based on sequence alignments and subunit connectivity. TTHA1264, an M16B protein found in Thermus thermophiles HB8, possesses an HXXEH motif essential for Zn2+ binding and catalytic activity. TTHA1265 is another member of M16B, which lacks the metal-binding motif but with a conserved active-site R/Y pair commonly found in the C-terminal half of M16 enzymes. Sequence analysis showed that two genes coding for TTHA1264 and TTHA1265 assemble into a single operon in the bacterial genome. Here, we report the crystal structure of TTHA1265 and TTHA1264/TTHA1265 complex from T. thermophilus HB8. Interestingly, when TTHA1264 and TTHA1265 are present alone, TTHA1264 forms a monomer, TTHA1265 forms a homodimer, respectively. However, TTHA264 and TTHA1265 assembled into a heterodimeric complex, indicating that they prefer to form heterodimer. Biochemical data further confirmed the heterodimeric assembly indicating intrinsic heterodimeric assembly of TTHA1264 and TTHA1265. This property of TTHA1264 and TTHA1265 is consistent with the characteristics of the M16B family.

The crystal structure of MreC provides insights into polymer formation.

MreC is a scaffold protein required for cell shape determination through interactions with proteins related to cell wall synthesis. Here, we determined the crystal structure of the major periplasmic part of MreC from Escherichia coli at 2.1 Å resolution. The periplasmic part of MreC contains a coiled-coil domain and two six-stranded barrel domains. The coiled-coil domain is essential for dimer formation, and the two monomers are prone to relative motion that is related to the small interface of ß-barrel domains. In addition, MreC forms an antiparallel filament-like structure along the coiled-coil direction, which is different from the helical array structure in Pseudomonas aeruginosa. Our structure deepens our understanding of polymer formation of MreC.

Two component system CpxR/A regulates the prodigiosin biosynthesis by negative control in Serratia marcescens FS14.

Prodigiosin is a tripyrrole red secondary metabolite synthesized by many microorganisms, including Serratia marcescens. In this study, we found that the deletion of the gene of sensor kinase CpxA dramatically decreased the prodigiosin production, while the deletion of the gene of the response regulator CpxR or both genes of CpxRA has no effect on prodigiosin production, the kinase function of CpxA is not essential for its regulation on prodigiosin production while the phosphorylation site of CpxR is required. We further demonstrated that the CpxA regulates the prodigiosin biosynthesis at the transcriptional level and the phosphatase activity of CpxA plays vital roles in the regulation of prodigiosin biosynthesis. Finally, we proposed that CpxR/A regulates the prodigiosin biosynthesis by negative control and the phosphorylation level of CpxR may determine the positive or negative control of the genes it regulated.

RpoS Activates the Prodigionsin Production by Activating the Transcription of the RpoS-Dependent Pig Gene Cluster in Serratia marcescens FS14.

RpoS, an alternative sigma factor of RNA polymerase, regulates the expression of a great deal of genes involved in stationary-phase survival and stress response. To identify the function of RpoS homologue in Serratia marcescens FS14, in-frame deletion mutant of rpoS was constructed. It was found that RpoS activates the biosynthesis of prodigiosin in FS14 which is just opposite to what was observed in Serratia sp. ATCC 39006. We also demonstrated that RpoS positively regulates the prodigiosin production by activating the transcription of pig cluster in FS14, and the transcription of pig cluster is RpoS-dependent. Further study showed that the differences in the promoters of pig clusters in FS14 and 39006 lead to the different selection of the sigma factors and result in the different regulation mechanisms. The -10 element and the spacer region between -10 and -35 elements of the pig cluster in FS14 are vital for the RpoS recognition in FS14. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12088-021-00952-4.

Protein-derived structures determines the redox capacity of humic acids formed during hyperthermophilic composting.

Humic acid (HA) in compost has received widespread attention for its high redox activity, which can mediate the degradation of organic pollution and the passivation of heavy metals in the environment. Hyperthermophilic composting (HTC) can accelerate HA formation. However, few studies have examined whether and how the structures of different organics affect the formation of the HA and HA redox structure at the molecular level in HTC. Detailed molecular information and the redox capacity (electron transfer capacity, ETC) of HA in HTC and thermophilic composting (TC) were characterized using pyrolysis gas chromatography/mass spectrometry and the electrochemical method, respectively. HTC promoted the formation of redox structure, leading to the improvement of the ETC of HA. Aromatics and N-containing compounds were mainly derived from protein components, and the rate at which they were transferred into HA was accelerated in HTC, while the relative abundance of lipids decreased. Partial least squares regression and correlation analysis demonstrated that protein-derived compounds were the key factor determining the HA redox capacity. Finally, partial least squares path modeling suggested that the influence mechanism of protein-derived structures on HA redox capacity might differ in HTC and TC. HTC may promote the relative abundance of N-containing components into the C-skeleton and accelerate the accumulation of the aromatic products, thereby improve the HA redox capacity. These findings provided new insight into how the redox capacity of the HA in compost could be improved and how compost products could be prepared for use in environmental remediation.

Evidence for the Rapid and Divergent Evolution of Mycoplasmas: Structural and Phylogenetic Analysis of Enolases.

Mycoplasmas are a group of prokaryotes without cell walls that have evolved through several rounds of degenerative evolution. With a low cell DNA G + C content and definitively long genetic lineages, mycoplasmas are thought to be in a state of rapid evolution. However, little associated evidence has been provided. Enolase is a key enzyme in glycolysis that is widely found in all species from the three domains, and it is evolutionarily conserved. In our previous studies, enolase acted as a virulence factor and participated in cell-surface adhesion in Mycoplasma hyopneumoniae. Furthermore, unique loop regions were first found in the crystal structure of Mhp Eno. Here, enolase structures from Mycoplasma pneumoniae and Mycoplasma bovis were determined. An extra helix 7 is specific and conservatively found in almost all mycoplasma enolases, as confirmed by crystal structures and sequence alignment. Particular motifs for helix 7, which is composed of F-K/G-K-L/F-K-X-A-I, have been proposed and could be regarded as molecular markers. To our surprise, the genetic distances between any two mycoplasma enolases were obviously longer than those between the two corresponding species themselves, indicating divergent evolution of mycoplasma enolases, whereas no horizontal gene transfer was detected in mycoplasma enolase genens. Furthermore, different evolutionary patterns were adopted by different loop regions of mycoplasma enolase. Enolases from different Mycoplasma species also showed different affinities for PLG and fibronectin. Our results indicate the rapid and divergent evolution of mycoplasma enolase and mycoplasmas. This study will also aid understanding the independent evolution of Mycoplasma species after separation from their common ancestor.